cisplatin-DNA Adduct Formation Research Articles

Glutathione S-transferase omega class 1 (GSTO1)-associated large extracellular vesicles are involved in tumor-associated macrophage-mediated cisplatin resistance in bladder cancer.

Bladder cancer poses a significant challenge to chemotherapy due to its resistance to cisplatin, especially at advanced stages. Understanding the mechanisms behind cisplatin resistance is crucial for improving cancer therapy. The enzyme glutathione S-transferase omega class 1 (GSTO1) is known to be involved in cisplatin resistance in colon cancer. This study focused on its role in cisplatin resistance in bladder cancer. Our analysis of protein expression in bladder cancer cells stimulated by secretions from tumor-associated macrophages (TAMs) showed a significant increase in GSTO1. This prompted further investigation into the role of GSTO1 in bladder cancer. We found a strong correlation between GSTO1 expression and cisplatin resistance. Mechanistically, GSTO1 triggered the release of large extracellular vesicles (EVs) that promoted cisplatin efflux, thereby reducing cisplatin-DNA adduct formation and enhancing cisplatin resistance. Inhibition of EV release effectively counteracted the cisplatin resistance associated with GSTO1. In conclusion, GSTO1-mediated EV release may contribute to cisplatin resistance caused by TAMs in bladder cancer. Strategies to target GSTO1 could potentially improve the efficacy of cisplatin in treating bladder cancer.

Abstract 1990: TIP60-mediated acetylation of ΔNp63α is associated with cisplatin resistance

Abstract The chemotherapeutic drug Cisplatin is often used to treat squamous cell carcinoma (SCC) patients, but low response rates and disease recurrence are common. About 5.4 million basal and squamous cell skin cancers are diagnosed each year in the US. ΔNp63α, a member of the p53 family of transcription factors, is overexpressed and considered oncogenic in non-melanoma skin cancer where it regulates cell survival, promotes proliferation, and inhibits cell apoptosis. ΔNp63α has also been shown to promote resistance to cisplatin by orchestrating the transcriptional regulation of several DNA damage response (DDR) genes. Our previous study showed that the histone acetyltransferase (HAT) TIP60 promotes SCC proliferation by positively regulating ΔNp63α protein levels in a manner reliant on the catalytic activity of TIP60. This observation suggested that TIP60 may contribute to the failure of platinum-based drugs in SCC and led us to hypothesize that TIP60-mediated acetylation of ΔNp63α regulates its stability and transcriptional activity to promote chemoresistance. We found that TIP60 levels positively correlated with ΔNp63α stability, protein levels, and cisplatin resistance. Further, silencing endogenous TIP60 led to a decrease in ΔNp63α transcript and protein levels in multiple cisplatin resistant SCC cell lines. Further, stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, whereas loss of ΔNp63α or TIP60 induced G2/M arrest, increased cell death, and sensitized cells to cisplatin. Additionally, pharmacological inhibition of TIP60 reduced acetylation of ΔNp63α, decreased colony formation, and sensitized resistant cells to cisplatin. Finally, we demonstrated that ΔNp63α and TIP60 levels positively correlated with DNA repair capacity and negatively correlated with cisplatin-DNA adduct formation. Silencing of either TIP60 or ΔNp63α intensified cisplatin-DNA adduct formation and significantly reduced the expression of drug efflux transporters. Taken together, our data indicate that TIP60-mediated stabilization of ΔNp63α increases cisplatin resistance and provides critical insights into the mechanisms by which ΔNp63α confers cisplatin resistance in SCC. Our findings suggest that the inhibition of TIP60 may be therapeutically advantageous in overcoming cisplatin resistance in SCC and other epithelial cancers. Citation Format: Akshay Hira, Jin Zhang, Mike Kemp, Madhavi P. Kadakia. TIP60-mediated acetylation of ΔNp63α is associated with cisplatin resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1990.

Abstract 400: TIP60-mediated regulation of DNp63a is associated with cisplatin resistance

Abstract About 5.4 million basal and squamous cell skin cancers are diagnosed each year in the US. The chemotherapeutic drug, Cisplatin is often used to treat squamous cell carcinoma (SCC) patients, but low response rates and disease recurrence is common. ΔNp63α, a member of the p53 family of transcription factors, is overexpressed and considered oncogenic in non-melanoma skin cancer where it regulates cell survival, promotes proliferation and inhibits cell apoptosis. ΔNp63α has also been shown to promote resistance to cisplatin by transcriptionally regulating several DNA damage response (DDR) genes. A previous study from our lab showed that the histone acetyltransferase (HAT) TIP60 promotes SCC proliferation by positively regulating ΔNp63α protein levels in manner dependent on the catalytic activity of TIP60. This finding suggested that TIP60 may contribute to the failure of platinum-based drugs in SCC and led us to hypothesize that TIP60-mediated acetylation of ΔNp63α regulates its stability and transcriptional activity to promote chemoresistance. Silencing endogenous TIP60 led to a decrease in ΔNp63α transcript and protein levels in multiple SCC cell lines, indicating the positive regulation of ΔNp63α by TIP60 is not cell-line specific. Further, TIP60 levels positively correlated with ΔNp63α stability, protein levels and cisplatin resistance. Stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, whereas loss of ΔNp63α and TIP60 induced G2/M arrest, increased cell-death and sensitized cells to cisplatin. Moreover, pharmacological inhibition of TIP60 reduced acetylation of ΔNp63α and sensitized resistant cells to cisplatin. Finally, we demonstrated that ΔNp63α and TIP60 levels positively correlated with DNA repair capacity and negatively correlated with cisplatin-DNA adduct formation. Silencing of either TIP60 or ΔNp63α enhanced cisplatin-DNA adduct formation and significantly reduced expression of genes involved in DDR. Taken together, our data indicate that TIP60-mediated stabilization of ΔNp63α increases cisplatin resistance and provides critical insights into the mechanisms by which ΔNp63α confers cisplatin resistance through regulation of genes involved in DNA damage repair. Our findings suggest that inhibition of TIP60 may be therapeutically advantageous in overcoming cisplatin resistance in SCC other epithelial cancers. Citation Format: Akshay Hira, Andrew J. Stacy, Jin Zhang, Mike Kemp, Madhavi P. Kadakia. TIP60-mediated regulation of DNp63a is associated with cisplatin resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 400.

The Molecular Basis and Therapeutic Aspects of Cisplatin Resistance in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a kind of malignant tumors with low survival rate and prone to have early metastasis and recurrence. Cisplatin is an alkylating agent which induces DNA damage through the formation of cisplatin-DNA adducts, leading to cell cycle arrest and apoptosis. In the management of advanced OSCC, cisplatin-based chemotherapy or chemoradiotherapy has been considered as the first-line treatment. Unfortunately, only a portion of OSCC patients can benefit from cisplatin treatment, both inherent resistance and acquired resistance greatly limit the efficacy of cisplatin and even cause treatment failure. Herein, this review outline the underlying mechanisms of cisplatin resistance in OSCC from the aspects of DNA damage and repair, epigenetic regulation, transport processes, programmed cell death and tumor microenvironment. In addition, this review summarizes the strategies applicable to overcome cisplatin resistance, which can provide new ideas to improve the clinical therapeutic outcome of OSCC.

A Label-free DNA-based Fluorescent Sensor for Cisplatin Detection

Cisplatin, an anti-cancer drug, has been used extensively and has become a “gold standard” treatment for various types of cancer. However, overdosage has serious side effects. Thus, this drug should not be over-administered. The current study presents a label-free but sensitive fluorescent sensor for cisplatin detection based on G-quadruplex (GQ) and fluorescent dye Thioflavin T (ThT). In the absence of cisplatin, ThT binds to GQ, resulting in high fluorescence emission. In the presence of cisplatin, the GQ structure is destroyed after cisplatin–DNA adduct formation, leaving no binding scaffold for ThT, thereby causing a decrease in ThT fluorescence. The amount of cisplatin can be quantified from the changes in ThT fluorescence intensity. Parameters that affect the sensor performance were tested, including pH and concentration of potassium phosphate buffer, type of GQ, number of quartets, loop length, incubation time, and temperature. The sensor provided a linear dynamic range of 10–500 nM with limit of quantification at 10 nM. The sensor was successfully applied to detect cisplatin in urine samples from patients undergoing treatment with high accuracy (recoveries between 74 ± 14 and 112.8 ± 5.0%) and precision (relative standard deviations between 1.15% and 2.12%).

Metformin enhances the radiosensitizing effect of cisplatin in non-small cell lung cancer cell lines with different cisplatin sensitivities

Cisplatin is an extensively used chemotherapeutic drug for lung cancer, but the development of resistance decreases its effectiveness in the treatments of non-small cell lung cancer (NSCLC). In this study, we examined the effects of metformin, a widely used antidiabetic drug, on cisplatin radiosensitization in NSCLC cell lines. Human NSCLC cell lines, A549 (cisplatin-resistant) and H460 (cisplatin-sensitive), were treated with metformin, cisplatin or a combination of both drugs before ionizing radiation. Cell proliferation, clonogenic assays, western blotting, cisplatin-DNA adduct formation and immunocytochemistry were used to characterize the treatments effects. Metformin increased the radiosensitivity of NSCLC cells. Metformin showed additive and over-additive effects in combination with cisplatin and the radiation response in the clonogenic assay in H460 and A549 cell lines (p = 0.018 for the interaction effect between cisplatin and metformin), respectively. At the molecular level, metformin led to a significant increase in cisplatin-DNA adduct formation compared with cisplatin alone (p < 0.01, ANOVA-F test). This was accompanied by a decreased expression of the excision repair cross-complementation 1 expression (ERCC1), a key enzyme in nucleotide excision repair pathway. Furthermore, compared with each treatment alone metformin in combination with cisplatin yielded the lowest level of radiation-induced Rad51 foci, an essential protein of homologous recombination repair. Ionizing radiation-induced γ-H2AX and 53BP1 foci persisted longer in both cell lines in the presence of metformin. Pharmacological inhibition of AMP-activated protein kinase (AMPK) demonstrated that metformin enhances the radiosensitizing effect of cisplatin through an AMPK-dependent pathway only in H460 but not in A549 cells. Our results suggest that metformin can enhance the effect of combined cisplatin and radiotherapy in NSCLC and can sensitize these cells to radiation that are not sensitized by cisplatin alone.

MiR-133b induces chemoresistance of osteosarcoma cells to cisplatin treatment by promoting cell death, migration and invasion.

As an important chemotherapeutic agent for the treatment of osteosarcoma, the effectiveness of cisplatin is considered to be due to its unique properties, which allow it to penetrate the cell membrane and form various DNA-platinum adducts, resulting in genetic alterations or DNA damage. However, chemoresistance to cisplatin remains a major challenge for its use and chemotherapeutic effects. In the present study, an isogenic model of a cisplatin resistant osteosarcoma cell line, MG63-DDP, was generated from the original MG63 cell line. The expression level of microRNA (miR)-133b in the MG63-DDP cisplatin-resistant osteosarcoma cell line was analyzed by reverse transcription-quantitative polymerase chain reaction (PCR). Cisplatin-DNA adduct formation, cell death (carboxyfluorescein succinimidyl ester/propidium iodide staining) and clonogenic survival assays (crystal violet staining) were performed, comparing various cell types. The effect of miR-133b on migration (scratch wound assay) and invasion (Transwell assay) was also evaluated. Characterization studies have previously revealed an increased level of miR-133b in MG63-DDP cells compared with normal MG63 cells. Upregulation of miR-133b was associated with the accumulation of cisplatin-DNA adducts and an increase in cisplatin-induced cell death. Furthermore, increased miR-133b expression levels enhanced the migration and invasion of MG63 cells under cisplatin stress. Concordantly, in MG63-DDP cells the neutralization of miR-133b demonstrated opposite effects, as compared with the upregulation of miR-133b. To the best of our knowledge, the present study demonstrated for the first time that cisplatin-resistant MG63 cells exhibit an increased level of miR-133b expression. The endogenous expression level of miR-133b is sufficient for inducing cisplatin resistance, which suggests that miR-133b may be a biomarker for cisplatin resistance in osteosarcoma.

Abstract 4682: Reversal of cisplatin resistance in human ovarian cancer through autophagy inhibition and caspase 1-α activation: Role of tumor associated Vacuolar-ATPase

Abstract The development of resistance to cisplatin drugs significantly hinders successful ovarian cancer treatment. An emerging centralized mechanism of chemo-resistance is a dysregulation in tumor cell pH (alkaline intracellular, acidic extracellular). Vacuolar-ATPase (V-ATPase) is a key proton pump that alters the pH and mediates metastasis/chemo-resistance in tumor cells. The major pH sensing unit of the V-ATPase complex is the ‘a2’ isoform(V0a2) and has been previously shown to promote metastasis in ovarian cancer cells. Here, we show that V0a2 is over-expressed in acquired cisplatin resistant ovarian tumor cells (cis-A2780 and cis-TOV112D) at both mRNA and protein levels. The shRNA mediated inhibition of V0a2 activity in cisplatin resistant cells (sh-V0a2-cis) sensitized the cells to cisplatin with a 3 fold reduction in inhibitory concentration 50% [IC50] (sh-V0a2-cis, IC50 = 11.47 ± 3.02 μM, p&amp;lt;0.05) compared to control resistant cells [IC50 = 37.75 ± 6.62 μM] as determined by Alamar blue cell cytotoxicity assay. Suppression of V0a2 activity strongly reduced the cytosolic pH in the resistant tumor cells (cis-A2780- pH = 7.19 ± 0.03; sh-V0a2-cis- pH = 6.87 ± 0.04; cisplatin sensitive cells- pH = 6.96± 0.01). This cytosolic acidification significantly enhanced DNA damage by cisplatin-DNA adduct formation as revealed by FACS and immuno-fluorescence analysis (p&amp;lt;0.05). To further define the cisplatin mediated multi-factorial responses in resistant cells upon V0a2 inhibition, we performed a PCR array for cell death pathway related genes. Several pro-apoptotic genes including caspase 3, caspase 9, Apaf-1, FASL, CD40,CD40L, TNF, TNFR1 were up-regulated (p&amp;lt;0.05). Notably, caspase-1α was significantly up-regulated upon cisplatin treatment in sh-V0a2-cis compared to control resistant cells (p&amp;lt;0.05). Caspase-1α is known to induce apoptosis of neurons and macrophages and is recently indicated to be pro-apoptotic in ovarian cancer cells. Additionally, several autophagy related genes such as LC3A, IGFR, ESR1, IRGM were also found up-regulated upon V0a2 inhibition. Impaired autophagy in sh-V0a2-cis was further confirmed at protein level by FACS analysis using autophagy detection kit. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 inhibits autophagy and activates caspase-1α upon cisplatin treatment in resistant ovarian cancer cells. V0a2 inhibition can therefore serve as a therapeutic strategy to overcome cisplatin resistance and in improving the treatment efficacy in chemo-resistant ovarian carcinoma. Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Sahithi Pamarthy, Alice Gilman-Sachs, Kenneth D. Beaman. Reversal of cisplatin resistance in human ovarian cancer through autophagy inhibition and caspase 1-α activation: Role of tumor associated Vacuolar-ATPase. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4682.

Abstract 3194: Cisplatin-DNA adduct formation in patients receiving cisplatin +/- sodium thiosulphate (STS) in the SIOPEL 6 randomized phase III trial

Abstract Background: The SIOPEL 6 randomised phase III trial was designed to investigate the efficacy of sodium thiosulphate (STS) in reducing ototoxicity in patients receiving cisplatin monotherapy for standard-risk hepatoblastoma. Preliminary results from the trial recently presented at ASCO indicated comparable response rates between the two study arms. The trial has now completed patient recruitment, with final evaluation of hearing loss at ≥3.5 years of age awaited as the primary endpoint of the trial. As part of the clinical study, blood samples were collected pre-treatment and 24 hours following cisplatin administration, to investigate the potential impact of STS on the formation of platinum-DNA adduct levels in leucocytes. Patients and methods: Whole blood samples (5-10ml) were taken prior to cisplatin treatment and 24 hours following the start of a 6 hour cisplatin infusion, with STS administered 6 hours after the end of cisplatin administration in patients randomised to receive STS. Samples were collected in EDTA blood tubes and frozen at -20°C or -80°C prior to transport to Newcastle for analysis. DNA was isolated from whole blood using Qiagen DNA blood Maxi kits and the concentration of DNA in each sample quantified by UV absorption. DNA samples were diluted in 3.5% nitric acid and hydrolyzed overnight at 70°C. Platinum-DNA adduct levels were determined by ICP-MS analysis, with results expressed in units of nmoles platinum/g DNA. Results: Blood samples were collected from a total of 37 patients, with 22/37 (59%) patients having received cisplatin followed by STS and 15/37 (41%) patients cisplatin alone. A significant difference in median platinum-DNA adduct levels was observed in patients receiving cisplatin followed by STS (median: 6.7 nmol/g DNA; range: 4.2 - 14.0), as compared to patients receiving cisplatin alone (median: 13.5 nmol/g DNA; range: 4.3 - 165.7) (P = 0.0011). Conclusion: Results indicate that the formation and/or longevity of platinum-DNA adducts determined in whole blood samples collected 24 hours following cisplatin administration are influenced by the co-administration of STS, with significantly higher levels observed in patients receiving cisplatin alone. Bearing in mind the comparable response data reported between the two randomisation study arms, these data would indicate that platinum-DNA adduct formation in leucocytes may act as a surrogate marker, providing novel insights into the mechanism of the otoprotective effects of STS when used in combination with cisplatin. Acknowledgements: Work supported by Cancer Research UK, the North of England Children's Cancer Research Fund and the Experimental Cancer Medicine Centre Network. Citation Format: Gareth J. Veal, Milind Ronghe, Bruce Morland, Edward Neuwelt, Rudolf Maibach, Penelope Brock. Cisplatin-DNA adduct formation in patients receiving cisplatin +/- sodium thiosulphate (STS) in the SIOPEL 6 randomized phase III trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3194.

Abstract 4560: Effects of cisplatin, vorinostat, and their combinations on EGFR-overexpressing cell lines

Abstract Cisplatin is one of the most commonly used drugs against human malignancies. Its cytotoxic effects are mainly mediated by formation of cisplatin-DNA adducts, which activate series of DNA damage-responsive proteins that ultimately trigger cell death. In spite of its potent activity, some malignancies treated with cisplatin may develop resistance after initial response while others may be intrinsically insensitive. Accordingly, cisplatin is frequently combined with other drugs to reduce resistance or to attain synergy. The presented work was conducted in EGFR-overexpressing cancer cells, DU-145 and MDA-MB-231, to examine the possible molecular effects of cisplatin alone or in combination with vorinostat, a histone deacetylase inhibitor. Proliferation assay, annexin V staining, western blotting and RT-PCR were used to study effects of those drugs on cell viability, apoptosis and molecular events in both cell lines. In the present study, incubation of DU-145 cells with cisplatin exerted dose-dependent antiproliferative and apoptosis-inducing effects. In addition, cisplatin significantly increased the apoptosis markers, cleaved PARP and cleaved caspase 7; depleted total EGFR and decreased ERK activation. This molecular response was also associated with dose-dependent increase in phosphorylated ATM (an early sensor of genotoxic stress), phosphorylated p38 and mRNA level of DDIT3 (a late sensor of DNA damage). On the other hand, in MDA-MB-231, cisplatin did not affect percent of apoptotic cells as compared with the untreated control, indicating a cisplatin-insensitive phenotype. The insensitive response towards cisplatin treatment was confirmed through absence of cleaved PARP and cleaved caspase 7 in total cell lysates. Although cisplatin increased the level of phosphorylated ATM and phosphorylated p38 in MDA-MB-231, it failed to affect DDIT3 mRNA level as compared with untreated cells. This may suggest that sensitivity towards cisplatin may be related to late DNA damage processing rather than early DNA damage response. Treating DU-145 and MDA-MB-231 cells with vorinostat elicited dose-dependent decrease in proliferation and increase in percentage of apoptotic cells. It also dose-dependently cleaved PARP and caspase 7, time-dependently degraded EGFR and decreased ERK activation in both cell lines. In spite of that potent activity, vorinostat synergized the effect of cisplatin only in DU-145 and failed to circumvent the insensitivity of MDA-MB-231 cells towards cisplatin. In conclusion, cisplatin decreased cell proliferation and induced apoptosis only in DU-145 cells but not MDA-MB-231 cells. Combination of vorinostat and cisplatin synergistically reduced percent cell viability and triggered apoptotic response in DU-145 cells when compared with the effects of individual drugs. However, in MDA-MB-231cells, addition of vorinostat to cisplatin did not significantly change any of the measured parameters. Citation Format: Asmaa Elsayed El-Kenawi, John Kenneth Cowell. Effects of cisplatin, vorinostat, and their combinations on EGFR-overexpressing cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4560. doi:10.1158/1538-7445.AM2014-4560

Metformin induces an intracellular reductive state that protects oesophageal squamous cell carcinoma cells against cisplatin but not copper-bis(thiosemicarbazones).

BackgroundOesophageal squamous cell carcinoma (OSCC) is a highly aggressive carcinoma with a poor survival rate. One of the most commonly used chemotherapeutic drugs, cisplatin, displays varied and often poor efficacy in vivo. Therefore, alternative, cost-effective and more efficacious treatments are required. Metformin has been previously shown to reduce proliferative rates in various carcinoma cell lines. We report for the first time, the effect of metformin on OSCC cell proliferation and show that it antagonises cisplatin-induced but not copper-bis(thiosemicarbazone)-induced cytotoxicity in OSCC cells.MethodsCell proliferation and stage of the cell cycle were quantified by trypan blue counts and flow cytometry, respectively. All cytotoxicity measurements were made using the tetrazolium based MTT assay. Metabolic alterations to cells were determined as follows: glycolysis via a lactate dehydrogenase assay, reducing equivalents by MTT reduction and reduced intracellular thiols by monobromobimane-thiol fluorescence, and glutathione depletion using buthionine sulfoximine. Inductively coupled plasma mass spectrometry was used to quantify cisplatin-DNA adduct formation.ResultsMetformin was found to reduce cell proliferation significantly in all OSCC cell lines, with an accumulation of cells in G0/G1 phase of the cell cycle. However, metformin significantly protected OSCC cells against cisplatin toxicity. Our results indicate that a major mechanism of metformin-induced cisplatin resistance results from a significant increase in glycolysis, intracellular NAD(P)H levels with a concomitant increase in reduced intracellular thiols, leading to decreased cisplatin-DNA adduct formation. The glutathione synthesis inhibitor buthionine sulfoximine significantly ablated the protective effect of metformin. We subsequently show that the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM, which are trapped in cells under reducing conditions, cause significant OSCC cytotoxicity, both alone and in combination with metformin.ConclusionsThis is the first study showing that metformin can be used to decrease cell proliferation in OSCC cells. However, metformin protects against cisplatin cytotoxicity by inducing a reducing intracellular environment leading to lower cisplatin-DNA adduct formation. As such, we advise that caution be used when administering cisplatin to diabetic patients treated with metformin. Furthermore, we propose a novel combination therapy approach for OSCC that utilises metformin with metformin-compatible cytotoxic agents, such as the copper-bis(thiosemicarbazones), Cu-ATSM and Cu-GTSM.

Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

IntroductionInherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin.MethodsAn isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed.ResultsCharacterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines.ConclusionOur results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer.

The interaction of cisplatin with a human telomeric DNA sequence containing seventeen tandem repeats

The anti-tumour drug, cisplatin, preferentially forms adducts at G-rich DNA sequences. Telomeres are found at the ends of chromosomes and, in humans, contain the repeated DNA sequence (GGGTTA)n that is expected to be targeted by cisplatin. Using a plasmid clone with 17 tandem telomeric repeats, (GGGTTA)17, the DNA sequence specificity of cisplatin was investigated utilising the linear amplification procedure that pin-pointed the precise sites of cisplatin adduct formation. This procedure used a fluorescently labelled primer and capillary electrophoresis with laser-induced fluorescence detection to determine the DNA sequence specificity of cisplatin. This technique provided a very accurate analysis of cisplatin-DNA adduct formation in a long telomeric repeat DNA sequence. The DNA sequence specificity of cisplatin in a long telomeric tandem repeat has not been previously reported. The results indicated that the 3′-end of the G-rich strand of the telomeric repeat was preferentially damaged by cisplatin and this suggests that the telomeric DNA repeat has an unusual conformation.

149P - Microrna Profiling and CDDP-GPG DNA Adduct Formation Analysis in a Panel of Cisplatin Resistant Non-Small Cell Lung Cancer Cell Lines

ABSTRACT Introduction The outcome of cisplatin therapy in NSCLC has reached a plateau, with the development of resistance being a major obstacle in the use of this drug. Understanding the molecular mechanisms underlying this resistance phenotype may aid in the development of novel agents that enhance the sensitivity of cisplatin. In this study, we examined the microRNA profile and levels of cisplatin-DNA adduct formation in a panel of cisplatin resistant NSCLC cell lines. Methods We have previously generated a panel of cisplatin resistant (CisR) cell lines (MOR, H460, A549, SKMES-1, H1299) from original, age-matched parent (PT) cells and extensively characterised these based on a number of functional assays including proliferation (MTT), apoptosis (FACS), cell cycle arrest (PI staining), survival ability (clonogenic assay), DNA copy number gains and losses (CGH arrays) and cancer stem cell marker expression (CD44, CD133, ALDH1, Nanog, Sox-2 and Oct-4) by FACS and Western blot analysis. MicroRNA profiling was carried out using the nCounter miRNA Expression Assay consisting of 749 target miRNA's. Immunofluorescence staining and measurement of specific DNA platination products (CDDP-GpG DNA adducts) was performed at 0, 4, 12 and 24h using immunofluorescence microscopy and quantitative digital image analysis using the ACAS 6.0 CytometryAnalysis System, respectively. Results In a panel of five NSCLC cell lines exhibiting a cisplatin resistant phenotype, a 3- and 13-miRNA signature was deduced, based on differential expression of 3 miRNA's (5/5 cell lines) and 13 miRNA's (4/5 cell lines) in the CisR cell lines relative to their parent counterparts. Levels of CDDP-GpG adducts were greatly reduced in resistant cells up to 24h in response to cisplatin compared to that observed in corresponding parent cells. Conclusion We have identified a distinct miRNA fingerprint in a clinically relevant, isogenic model of cisplatin resistance that may predict resistance to cisplatin in lung cancer. Furthermore, as cisplatin-DNA adducts correlate with cisplatin resistance in vitro, these may be used as a potential predictive marker in patient response to platinum therapy in NSCLC. Disclosure All authors have declared no conflicts of interest.

Abstract 1814: Claudin-3 and claudin-4 regulate sensitivity to cisplatin by controlling expression of the copper and cisplatin influx transporter CTR1

Abstract Cell adhesion complexes play a central role in controlling the behavior of epithelial cells, and loss of cell-cell adhesion is a characteristic of virtually all epithelial malignancies. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are the major structural molecules that form tight junctions between epithelial cells. We found that knockdown of the expression of either CLDN3 or CLDN4 produced marked changes in the phenotype of ovarian cancer cells including an increase in in vivo growth rate and metastatic potential. However, the role of claudins in controlling sensitivity of ovarian cancer cells to the platinum-containing drugs, and the mechanisms by which they regulate the sensitivity, remain largely unknown. The effect of CLND3 and CLDN4 on cisplatin cytotoxicity, cellular accumulation and DNA adduct formation was compared in the CLDN3- and CLDN4-expressing parental human ovarian carcinoma 2008 cells and CLDN3 or CLDN4 knockdown sublines (CLDN3KD and CLDN4KD, respectively). CLDN3KD cells were 3.1-fold resistant to cisplatin whereas CLDN4KD cells were 8.0-fold resistant compared to the parental 2008 cells. The net accumulation of platinum in CLDN3KD and CLDN4KD cells was reduced to 66% and 49% of that in the parental cells after exposure to 30 μM cisplatin for 1 h. Likewise, the DNA adduct levels dropped to 65% and 41% of control when CLDN3 and CLDN4 were knocked down. Given that transporters and chaperones that manage copper homeostasis regulate platinum drug accumulation and efflux, their expression was measured by qRT-PCR. Among these, only the expression of CTR1 was significantly reduced (p&amp;lt;0.01) in the knockdown cells while there was no appreciable change in CTR2, ATP7A, ATP7B or ATOX1 mRNA levels. Consistent with the decrease in CTR1 level, the average steady-state copper level in the 2008 cells was 6.4 ± 1.26 ng Cu/μg S whereas the CLDN3KD and CLDN4KD cells contained only 4.2 ± 0.87 and 4.0 ± 0.74 ng Cu/μg S resulting in a 34% (p=0.008) and 38% (p=0.004), respectively, loss of intracellular copper. Copper-dependent tyrosinase activity was also markedly reduced in those CLDN knockdown cells when incubated with the substrate L-DOPA. These results indicate that CLDN3 and CLDN4 affect sensitivity to the cytotoxic effect of cisplatin by regulating expression of the copper transporter CTR1 that we have previously shown to play a central role in the influx of cisplatin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1814. doi:1538-7445.AM2012-1814

On the complexity and dynamics of in vivo Cisplatin–DNA adduct formation using HPLC/ICP-MS

In this work we present a methodology to measure the complex adduct spectrum caused by the interaction of Cisplatin with DNA. By using an optimized DNA digestion procedure we were able to show that the adduct spectrum in in vivo duplex DNA is much more complex than described so far. For the first time a high abundance of interstrand adducts has been detected by using HPLC/ESI-MS. These adducts could play a key role in the DNA repair mechanisms and the development of cellular resistance to Cisplatin. By species-unspecific isotope dilution analysis HPLC/ICP-MS measurements, we were able to study the kinetics of adduct formation. With these experiments we proved that after the initial formation of adducts a rearrangement occurs on the DNA-strands leading to significant changes in adduct patterns over time. Furthermore, the parameters of the species-unspecific isotope dilution analysis were optimized to allow measurements of specific adducts in the DNA of Cisplatin exposed cells.

Metallofullerene nanoparticles circumvent tumor resistance to cisplatin by reactivating endocytosis

Cisplatin is a chemotherapeutic drug commonly used in clinics. However, acquired resistance confines its application in chemotherapeutics. To overcome the acquired resistance to cisplatin, it is reasoned, based on our previous findings of mediation of cellular responses by [Gd@C(82)(OH)(22)](n) nanoparticles, that [Gd@C(82)(OH)(22)](n) may reverse tumor resistance to cisplatin by reactivating the impaired endocytosis of cisplatin-resistant human prostate cancer (CP-r) cells. Here we report that exposure of the CP-r PC-3-luc cells to cisplatin in the presence of nontoxic [Gd@C(82)(OH)(22)](n) not only decreased the number of surviving CP-r cells but also inhibited growth of the CP-r tumors in athymic nude mice as measured by both optical and MRI. Labeling the CP-r PC-3 cells with transferrin, an endocytotic marker, demonstrated that pretreatment of the CP-r PC-3-luc cells with [Gd@C(82)(OH)(22)](n) enhanced intracellular accumulation of cisplatin and formation of cisplatin-DNA adducts by restoring the defective endocytosis of the CP-r cancer cells. The results suggest that [Gd@C(82)(OH)(22)](n) nanoparticles overcome tumor resistance to cisplatin by increasing its intracellular accumulation through the mechanism of restoring defective endocytosis. The technology can be extended to other challenges related to multidrug resistance often found in cancer treatments.

Cisplatin sensitivity is related to late DNA damage processing and checkpoint control rather than to the early DNA damage response

The present study aimed at elucidating mechanisms dictating cell death triggered by cisplatin-induced DNA damage. We show that CL-V5B hamster mutant cells, a derivative of V79B, are hypersensitive to cisplatin-induced apoptotic death. CL-V5B cells are characterized by attenuated cisplatin-induced early (2–6 h) stress response, such as phosphorylation of stress-activated protein kinases (SAPK/JNK), ATM and Rad3-related (ATR) protein kinase, histone H2AX and checkpoint kinase-1 (Chk-1). Human FANCC cells also showed a reduced phosphorylation of H2AX and SAPK/JNK at early time point after cisplatin treatment. This was not the case for BRCA2-defective VC-8 hamster cells, indicating that the FA core complex, rather than its downstream elements, is involved in early damage response. The alleviated early response of CL-V5B cells is not due to a general dysfunction in ATM/ATR-regulated signaling. It is rather due to a reduced formation of primary cisplatin-DNA adducts in the hypersensitive mutant as shown by analysis of DNA platination, DNA intra- and interstrand crosslink formation and DNA replication blockage. Despite of lower initial DNA damage and attenuated early DNA damage response (DDR), CL-V5B cells are characterized by an excessive G2/M arrest as well as an elevated frequency of DNA double-strand breaks (DSB) and chromosomal aberrations (CA) at late times (16–24 h) after cisplatin exposure. This indicates that error-prone processing of cisplatin-induced lesions, notably interstrand crosslinks (ICL), and the formation of secondary DNA lesions (i.e. DSB), results in a powerful delayed DNA damage response and massive pro-apoptotic signaling in CL-V5B cells. The data provide an example that the initial level of cisplatin-DNA adducts and the corresponding early DNA damage response do not necessarily predict the outcome of cisplatin treatment. Rather, the accuracy of DNA damage processing and late checkpoint control mechanisms determine the extent of cell death triggered by cisplatin-induced DNA lesions.

Cisplatin-DNA adduct formation in patients treated with cisplatin-based chemoradiation: lack of correlation between normal tissues and primary tumor

PurposeIn this study, the formation of cisplatin-DNA adducts after concurrent cisplatin-radiation and the relationship between adduct-formation in primary tumor tissue and normal tissue were investigated.MethodsThree intravenous cisplatin-regimens, given concurrently with radiation, were studied: daily low-dose (6 mg/m2) cisplatin, weekly 40 mg/m2, three-weekly 100 mg/m2. A 32P-postlabeling technique was used to quantify adducts in normal tissue [white blood cells (WBC) and buccal cells] and tumor.ResultsNormal tissue samples for adduct determination were obtained from 63 patients and tumor biopsies from 23 of these patients. Linear relationships and high correlations were observed between the levels of two guanosine- and adenosine–guanosine-adducts in normal and tumor tissue. Adduct levels in tumors were two to five times higher than those in WBC (P < 0.001). No significant correlations were found between adduct levels in normal tissues and primary tumor biopsies, nor between WBC and buccal cells.ConclusionsIn concurrent chemoradiotherapy schedules, cisplatin adduct levels in tumors were significantly higher than in normal tissues (WBC). No evidence of a correlation was found between adduct levels in normal tissues and primary tumor biopsies. This lack of correlation may, to some extent, explain the inconsistencies in the literature regarding whether or not cisplatin-DNA adducts can be used as a predictive test in anticancer platinum therapy.

Prediction of treatment outcome by cisplatin‐DNA adduct formation in patients with stage III/IV head and neck squamous cell carcinoma, treated by concurrent cisplatin‐radiation (RADPLAT)

The purpose of our study was to test the predictive value of cisplatin-DNA adduct levels in head and neck squamous cell carcinoma (HNSCC) patients treated with cisplatin-radiation. Patients with advanced-stage HNSCC were treated within a randomized trial, investigating the optimal route of cisplatin administration, concurrently with radiation. Cisplatin was administered intra-arterially (IA, 150 mg/m2, with systemic rescue by sodium thiosulfate) or intravenously (IV, 100 mg/m2). In a subgroup, adducts were quantified in normal tissue and tumor. 32P-postlabeling was used to quantify intrastrand guanosine-guanosine adducts (GG-adducts) and adenosine-guanosine adducts (AG-adducts). Adduct levels were correlated with treatment outcome. Thirty-five patients were included (21 IV and 14 IA). At median follow-up of 27 months, locoregional (LR) control was 75% at 1 and 70% at 2 years. Adduct levels in tumor were 4-5-fold higher than in white blood cells (WBC) for both IA and IV treatment (p = 0.01). Adduct formation in WBC and buccal cells was higher in IV treated patients compared with IA infusion (p = 0.049 and 0.005 for GG-adducts in WBC and buccal cells, respectively). Adducts in tumors after IA infusion were not statistically different from those after IV. A strong correlation was observed between GG- and AG-adduct formation (r = 0.86, p < 0.001). Patients with higher GG adduct levels (>median) in primary tumor had significantly better disease free survival (DFS) than patients with lower (< or = median) adduct levels (p = 0.02). For overall survival (OS), a nonsignificant trend was observed, again in favor of patients with higher adduct levels (p = 0.06). In conclusion, cisplatin-DNA adduct formation in primary tumor appears to be predictive for DFS in HNSCC. No differences were observed in intratumoral adduct levels between IA and IV treatments, despite selective infusion of high-dose cisplatin with the IA procedure. However, systemic adduct levels (WBC and buccal cells) from IV patients were higher than in IA patients, consistent with less systemic exposure after IA administration.